Figure 1

PC12 cells were treated with 1 mM cocaine (COC) or PBS for 6 hours. Nuclear extract (5 μg) was submitted to EMSA assay. (A) Positions of protein/DNA complexes observed are indicated by numbers. Localization of free probe is also indicated. (B) Competition studies in the absence or presence of unlabeled specific (NF-κB consensus sequence 2-fold molar in excess) or non-specific oligonucleotide (TFIID consensus sequence, 2-fold molar in excess), as indicated. (C) Supershift assays incubated in the absence or presence of 2 μL of antibodies (1:10 dilution) against p50, p65, c-Rel and p52 subunits, as indicated. Antibodies were added 20 min prior to addition of the radiolabeled NF-κB consensus oligonucleotide. The position of specific NF-κB/DNA binding complex p50/p65 (band 1) is indicated. NS represents no specific binding (bands 2 and 3). (D) Concentration response curve and time-course of NF-κB activation induced by COC in PC12 cells. Nuclear proteins (5 μg) were extracted from cells treated with different concentrations of cocaine: 0.5, 0.25,0.5, 0.75, 1, 2 mM or PBS for 6 hours (E) Different time points of NF-κB activation: 1, 3, 6 and 8 hours with 1 mM of COC (panel B) or (F) 24 hours with 1 and 2 mM COC or PBS. The position of specific NF-κB/DNA binding complex (p50/p65 complex) is indicated. (G) Representative immunoblot expression of IκB-α in PC12 cells after incubation with COC (1 mM) or PBS for 6 hours. The densitometric analysis (% of control) of the IκB-α band complex represented in (G) is shown (H). β-Actin was used as internal control. Results are expressed as mean + S.E.M. from three individual experiments. * p < 0.05 vs PBS (Student's t-test).